reply to "Successful backcrosses" query

Terry Delaney DELANEYT at A1.CG.COM
Thu Sep 24 14:38:00 EST 1992


In response to Lee Meisel at the Waksman Institute, who asks about the
technical aspects of performing backcrosses and crosses with Arabidopsis..

I am posting this to the net in case anyone elso is interested..

        > If anyone out there in Arabidopsis land could supply me with technical         
        > information (published or unpublished) on crosses it would be very 
        >beneficial....I am [presently] performing the crosses under a  
        >mechanical drawing lamp...

I have a fair number of Arab crosses, and it can be a pain but it work reliably 
with practice.  I prefer to dissect flowers under a dissecting scope.  Some 
people like the head mounted magnifying goggles.  Unless the lens you are 
working over is greater than 10 or 20x mag, I think it may be difficult to 
monitor your work adequately; -it would be for me.  If possible, choose a 
phenotypically marked female line (i.e. homoz recess), and then you can easily 
discern the F1s from plants created by self pollination of the female.  Use very
fine forceps, free from drops on the table, and don't flame sterilize.  
Otherwise the tips aren't perfect.

I select flowers to act as pollen recipients, as those that have not yet pushed 
their stigma into view through sepals.  Once you can see the stigma, there is a 
fair chance that the flower has already self-pollinated.  For pollen donors, I 
select flowers where the petals are open, but not yet perpendicular to the main 
axis of the flower.  Under a 'scope, you can easily see whether the stigmatic 
hairs are glisteny and receptive, and whether the pollen is abundant and not 
dried out.  You can also see when lots of pollen has been transferred in the 
cross.  Pick one or a few flowers, remove their neighbors, and dissect the 
selected flower.  Narrow tape strips or thread can work well to label things. 

I pollinate the flowers at the same time as the dissection.  I have heard of 
waiting a day or two, but I would worry about dessication of the flower.  I 
can't speak from experience here, however.  If others have an alternate view 
here, please drop a note into the bucket.

        >is it necessary to pollenate the emassculated carpels early in the 
        >morning to increase your success rate?

I've not heard of any advantage over when in the day you do your crosses.  
Perhaps you may want to work early in the day (before you drink too much 
coffee!).  

re:  Environment... prior to fooling around with your plants, don't worry about 
it.. just get them when they're happy and healthy and the flowers are 
appropriate in age.  After the crosses, baby the plants a bit, a growth chamber 
helps if its available.  Greenhouses or benchtops will do if the female parental
line is not too wounded (genetically).  If your cross was successful you will 
see the style elongate within a day or two.  Be sure to keep the adjacant flower
buds pruned, or at least mark the flower of interest well to avoid later 
confusion.  When the silique is fairly dry, remove it and dry the pod for a week
or so prior to planting the seeds.  If you are in a hurry, you may add some 
drierite to the vial, and upon sowing give the seeds a night otr two at 5 
degrees C to aid germination.  Examine the F1 for any female parental recessive 
phenotype, and if seen discard that plant, since it has resulted from selfing.

Thats enough, good luck.

Terry Delaney
Department of Molecular Genetics
CIBA-GEIGY Ag Biotech
P.O. Box 12257
Research Triangle Park, NC 27709-2257
Telephone 919 541 8500 ext 8577
Telefax 919 541 8557
e-mail: delaneyt at a1.cg.com



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